Combination therapy with wt1 peptide vaccine and temozolomide

ABSTRACT

The present invention relates to combination therapy for cancer with a WT1 peptide vaccine and temozolomide.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority based on U.S. Provisional ApplicationNo. 61/552,209, filed Oct. 27, 2011 (incorporated herein by reference inits entirety).

TECHNICAL FIELD

The present invention relates to combination therapy for cancer with aWT1 peptide vaccine and temozolomide. The present invention also relatesto a pharmaceutical composition comprising a WT1 peptide, characterizedby being used in combination with temozolomide.

BACKGROUND ART

WT1 gene (Wilms' tumor 1 gene) is a gene identified as a causative genefor Wilms' tumor that is a childhood renal cancer, and encodes atranscription factor having a zinc finger structure. WT1 gene wasinitially considered to be a tumor suppressor gene. However, subsequentstudies revealed that it rather functioned as an oncogene in ahematopoietic organ tumor and a solid cancer.

It has been shown that the in vitro stimulation of peripheral bloodmononuclear cells with a WT1 peptide induces cytotoxic T-lymphocytes(CTLs) specific for the peptide, which kill hematopoietic organ tumor orsolid cancer cells endogenously expressing WT1. Various WT1 peptidescapable of inducing WT1-specific CTLs, which are expected to be used forcancer immunotherapy, have previously been identified (PatentLiteratures 1 to 11). Phase I and phase II clinical trials for cancerimmunotherapy using WT1 peptides have also been conducted (Non PatentLiteratures 1 to 4).

The presence of helper T cells specific for a cancer antigen is reportedto be important for the efficient induction of CTLs. Helper T cells areinduced and activated by recognizing a complex of an MHC class IImolecule in antigen-presenting cells and an antigen peptide to therebypromote the proliferation, differentiation, and maturation of B cellsand subsets of other T cells. Therefore, it is considered to be usefulin cancer immunotherapy to activate the immune system through theinduction of helper T cells by an antigen peptide that binds to an MHCclass II molecule; a plurality of WT1 peptides having such a functionhave also previously been reported (Patent Literatures 12 to 14 and NonPatent Literature 5).

Gliomas are primary brain tumors arise from glial cells. What areconsidered to be malignant among gliomas are glioblastoma and anaplasticastrocytoma, and glioblastoma is a tumor occurring most frequently andhaving high malignancy among primary brain tumors and is apoor-prognosis disease having a life expectancy of 12 to 14 months and a5-year survival of 5%. Surgery is the first choice for the treatment ofnewly-diagnosed malignant glioma; however, it is standard therapytherefor to perform radiation therapy and the administration oftemozolomide as a chemotherapeutic agent against postoperative residualtumor cells because the glioma shows a marked tendency for invasion andis difficult to completely remove. However, its therapeutic results arenot satisfactory with a median overall survival (OS) of 14.6 months, amedian progression-free survival (PFS) of 6.9 months, and a median2-year survival of 26% (Non Patent Literature 6).

Most chemotherapeutic agents nonspecifically affect not only tumor cellsbut also normal cells at a proliferative phase, and lymphocytesproliferated and activated by antigen stimulation are also similarlyaffected. Therefore, the immune system of a patient receivingchemotherapy is considered to be suppressed; regarding the simultaneouscombined use of chemotherapy and immunotherapy, concerns exist aboutdeleterious effects of chemotherapy on CTLs, i.e., cell-killing effectson CTLs in addition to the same effects on cancer cells. No therapyinvolving the combined use of cancer peptide vaccine therapy andtemozolomide has previously been reported.

CITATION LIST Patent Literature

-   [Patent Literature 1] International Publication No. WO 2000/006602-   [Patent Literature 2] International Publication No. WO 2002/079253-   [Patent Literature 3] International Publication No. WO 2003/106682-   [Patent Literature 4] International Publication No. WO 2004/026897-   [Patent Literature 5] International Publication No. WO 2004/063217-   [Patent Literature 6] International Publication No. WO 2005/095598-   [Patent Literature 7] International Publication No. WO 2007/097358-   [Patent Literature 8] International Publication No. WO 2008/081701-   [Patent Literature 9] International Publication No. WO 2009/072610-   [Patent Literature 10] International Publication No. WO 2007/063903-   [Patent Literature 11] International Publication No. WO 2000/018795-   [Patent Literature 12] International Publication No. WO 2008/105462-   [Patent Literature 13] International Publication No. WO 2010/123065-   [Patent Literature 14] International Publication No. WO 2005/045027

Non Patent Literature

-   [Non Patent Literature 1] Proc. Natl. Acad. Sci. USA, 2004; 101(38):    13885-90-   [Non Patent Literature 2] Int. J. Hematol., 2003; 78(1): 56-61-   [Non Patent Literature 3] Jpn. J. Clin. Oncol., 2006 April; 36(4):    231-6-   [Non Patent Literature 4] J. Neurosurg., 2008 May; 108 (5): 963-71-   [Non Patent Literature 5] Cancer. Immunol. Immunother., 51:271, 2002-   [Non Patent Literature 6] N. Engl. J. Med., 2005 Mar. 10; 352(10):    987-96

The above literatures are incorporated herein by reference.

SUMMARY OF INVENTION

An object of the present invention is to provide a more effective methodfor treating cancer using a WT1 peptide vaccine.

The present inventor has found that chemotherapy with temozolomide isused in combination with WT1 peptide vaccine therapy in malignant gliomapatients after extirpative surgery for tumor to provide an effectiveanti-tumor effect and prolong survival, thereby accomplishing thepresent invention.

Thus, the present invention provides a method for treating cancer,comprising administering therapeutically effective doses of a WT1peptide and temozolomide to a patient in need thereof.

The present invention also provides a pharmaceutical compositioncomprising a WT1 peptide for treating cancer, wherein the composition isused in combination with temozolomide. The present invention furtherprovides a pharmaceutical composition comprising temozolomide fortreating cancer, wherein the composition is used in combination with aWT1 peptide.

The present invention also provides a kit for treating cancer,comprising a WT1 peptide and temozolomide.

The present invention also provides use of a WT1 peptide for treatingcancer, wherein the WT1 peptide is used in combination withtemozolomide.

According to the present invention, the combination of cancerimmunotherapy targeting WT1 with chemotherapy using temozolomide enablesmore effective therapy of cancer than WT1 alone or temozolomide alone.

DESCRIPTION OF EMBODIMENTS

The present invention relates to a method for treating cancer,comprising administering therapeutically effective doses of a WT1peptide and temozolomide to a patient in need thereof.

The method of the present invention is characterized in that cancerimmunotherapy with a WT1 peptide vaccine that induces or potentiates thekilling of WT1-expressing cancer cells is used in combination withtemozolomide as a chemotherapeutic agent. The WT1 peptide according tothe present invention may be a peptide capable of binding to an MHCclass I molecule to induce antigen-specific CTLs (hereinafter, referredto as a CTL peptide), or a peptide capable of binding to an MHC class IImolecule to induce antigen-specific helper T cells (hereinafter,referred to as a helper peptide).

Specifically, the WT1 peptide of the present invention is a peptideconsisting of a portion of the amino acid sequence of human WT1 proteinshown in SEQ ID NO: 1, capable of binding to an MHC class I molecule toinduce WT1-specific CTLs (i.e., having a function as the CTL peptide),or a peptide consisting of an amino acid sequence in which one orseveral, preferably one or two amino acids are substituted, deleted,and/or added in the amino acid sequence of the above peptide, having afunction as the CTL peptide. The WT1 peptide may also be a peptideconsisting of a portion of the amino acid sequence of human WT1 proteinof SEQ ID NO: 1, capable of binding to an MHC class II molecule toinduce WT1-specific helper T cells (i.e., having a function as thehelper peptide), or a peptide consisting of an amino acid sequence inwhich one or several, preferably one or two amino acids are substituted,deleted, and/or added in the amino acid sequence of the above peptide,having a function as the helper peptide.

The WT1 peptide is not particularly limited in the amino acid sequenceand length thereof provided that it has the above characteristics. TheCTL peptide preferably consists of 6 to 30 amino acids, more preferably7 to 20 amino acids, still more preferably 8 to 12 amino acids, forexample, 8, 9, 10, or 11 amino acids, considering binding properties tothe MHC class I molecule. The helper peptide preferably consists of 10to 25 amino acids, more preferably 15 to 21 amino acids, still morepreferably 16 to 20, for example, 17, 18, or 19 amino acids, consideringbinding properties to the MHC class II molecule.

The MHC class I molecule to which the peptide of the present inventionbinds may be any subtype thereof, and examples thereof include HLA-A02,HLA-A11, HLA-A24, HLA-A26, and HLA-A33. The MHC class II molecule towhich the peptide of the present invention binds may also be any of thesubtypes of HLA-DR, HLA-DQ, and HLA-DP.

The amino acid substitution may be performed between any amino acids atany position in the amino acid sequence of the peptide; however, it ispreferably a conservative amino acid substitution. For example, it ispreferable to substitute an Asp residue for a Glu residue, a Tyr residuefor a Phe residue, a Ile residue for a Leu residue, a Ser residue forAla residue, or an Arg residue for a His residue. The addition anddeletion of an amino acid may be performed at any positions; however,they are preferably performed at the N terminal end and C terminal endof the peptide.

When the sequence regularities (motifs) of the antigen peptide bindingto the MHC class I and class II molecules are revealed, the amino acidsubstitution is preferably performed based on the motifs. For example,in the case of HLA-A24, it is known that the amino acid at the secondposition of a peptide consisting of 8 to 11 amino acids is tyrosine,phenylalanine, methionine, or tryptophan and the C-terminal end thereofis phenylalanine, leucine, isoleucine, tryptophan, or methionine (J.Immunol., 152, p3913, 1994; Immunogenetics, 41: p178, 1995, J. Immunol.,155: p4307, 1994) (both are incorporated herein by reference). Thus, inthe case of a HLA-24-binding peptide, amino acids at the second positionand/or at the C-terminal end are preferably substituted with amino acidswithin the above-described range. This is ditto for other subtypes ofthe MHC class I and class II molecules such as HLA-A2 (Immunogenetics,41, p178, 1995; J. Immunol., 155: p4749, 1995) (both are incorporatedherein by reference) and HLA-DRB1*0405 (Immunogenetics, 41, 178-228(1995); Biochimica et Biophysica Acta 1316, 85-101, 1996) (both areincorporated herein by reference).

The amino acid sequences of peptides predicted to be capable of bindingto MHC class I and class II molecules can be searched using BIMASsoftware of NIH on the Internet(http://bimas.dcrt.nih.gov/molbio/hla_bind/), BIMAS HLA peptide bindingprediction analysis (J. Immunol., 152, 163, 1994), the MHC classII-binding sequence prediction program, ProPred, on the Internet(Bioinformatics 17: 1236, 2001), or the like.

The W1 peptide may have one or a plurality of modified amino acidresidues. Examples of the modification include the esterification,alkylation, halogenation, phosphorylation, sulfonation, and amidation ofan amino acid residue. The WT1 peptide may also be modified bycondensing a thiol group of a cysteine residue and a thiol group ofcysteine, glutathione, thioglycolic acid, or the like, in the peptidevia disulfide bonding (see International Publication No. WO 2007/063903(incorporated herein by reference)). In addition, the WT peptide may beused in the form of a peptide dimer in which two monomers of the peptideare bonded together via disulfide bonding between the thiol groups of atleast one pair of cysteine residues between the two monomers (seeInternational Publication No. WO 2004/063217 (incorporated herein byreference)). The amino acids constituting the WT1 peptide may be naturaloccurring amino acids or synthetic amino acids. Such peptidemodification is only illustrative, and the peptides modified by methodsknown in the art can be all used in the present invention.

The WT1 peptide can be synthesized by methods usually used in the art ormodifications thereof. Such synthesis methods are described, forexample, in Peptide Synthesis, Interscience, New York, 1966; TheProteins, Vol. 2, Academic Press Inc., NewYork, 1976; “Pepuchido Gosei(Peptide Synthesis)”, Maruzen Co., Ltd., 1975; Pepuchido Gosei No KisoTo Jikken (Basis and Experiment for Peptide Synthesis) published byMaruzen Co., Ltd., 1985; and “Pepuchido Gosei (Peptide Synthesis)”,Iyakuhin No Kaihatsu Zoku (Development of Pharmaceutical Product(Continued)) vol. 14, published by Hirokawa Shoten, 1991 (all areincorporated herein by reference). The WT1 peptide can also be producedusing genetic engineering procedures on the basis of the information ofthe nucleotide sequence encoding the peptide. Such genetic engineeringprocedures are well known to those skilled in the art and are described,for example, in Molecular Cloning, T. Maniatis et al., CSH Laboratory(1983); DNA Cloning, DM. Glover, IRL PRESS (1985) (both are incorporatedherein by reference).

Whether the peptide has a function as the CTL peptide can be examined bya method as described, for example, in J. Immunol., 154, p2257, 1995(incorporated herein by reference). Specifically, when peripheral bloodmononuclear cells are isolated from an HLA antigen-positive human andstimulated by adding a candidate peptide in vitro, CTLs specificallyrecognizing HLA-positive cells pulsed with the peptide can be induced toconfirm that the peptide has antigen-specific T cell-inducing activity.Here, the presence of CTL induction can be examined, for example, bymeasuring the amount of IFN-γ produced by CTLs in response toantigen-presenting cells presenting the peptide by an enzyme-linkedimmunosorbent assay (ELISA). The examination can also be carried out bymeasuring the amount of TNF-α produced by CTLs in response toantigen-presenting cells, based on the survival rate of aTNF-α-sensitive cell line (for example, WEHI164S cells; ATCC No.CRL-1751). In addition, the examination can also be performed by amethod for measuring the killing property of CTLs againstantigen-presenting cells labeled with ⁵¹Cr (⁵¹Cr release assay, Int. J.Cancer, 58: p317, 1994 (incorporated herein by reference)). Theexamination can also be examined by pulsing cells in which an expressionplasmid capable of expressing cDNA for HLA is introduced e.g., intoCOS-7 cells (ATCC No. CRL1651) or VA-13 cells (Riken Cell Bank), with acandidate peptide, reacting these cells with the CTLs prepared above andthe like, and measuring the amount of various cytokines (e.g., IFN-γ andTNF-α) produced by the CTLs.

Whether the peptide has a function as the helper peptide can be examinedby a known method as described in Cancer Immunol. Immunother. 51:271,2002 (incorporated herein by reference) or the like. For example,dendritic cells (adherent cells) are prepared by recovering peripheralblood mononuclear cells from an HLA antigen-positive human and removingfloating cells therefrom. Separately, helper T cells (CD4-positive Tcells) are prepared from the same HLA antigen-positive human by aFicoll-Paque density gradient centrifugation method. Then, a candidatepeptide is added to the above-described dendritic cells, which is thencultured, followed by mix-culturing the dendritic cells and theabove-described helper T cells. Thereafter, the helper T cells arerecovered and stimulated a number of times with the dendritic cellspulsed with the candidate peptide. The induction (activation) of helperT cells in response to the peptide stimulation can be examined, forexample, by measuring (1) the growth activity of helper T cells or (2)the cytokine-producing-activity of helper T cells. The growth activityin (1) can be examined by measuring the amount of [³H]-thymidineincorporated into the helper T cells. The cytokine-producing-activity in(2) can be examined by measuring the amount of cytokines such as IFN-γproduced by activated helper T cells by ELISA or the like.

Examples of the WT1 peptide suitable for the present invention includeWT1 peptides as described in the following references: InternationalPublication No. WO 2000/006602; International Publication No. WO2002/079253; International Publication No. WO 2003/106682; InternationalPublication No. WO 2004/026897; International Publication No. WO2004/063217; International Publication No. WO 2005/095598; InternationalPublication No. WO 2007/097358; International Publication No. WO2008/081701; International Publication No. WO 2009/072610; InternationalPublication No. WO 2007/063903; International Publication No. WO2000/018795; International Publication No. WO 2008/105462; InternationalPublication No. WO 2010/123065; International Publication No. WO2005/045027; Cancer. Immunol. Immunother. 51:271, 2002 (PatentLiteratures 1 to 13 and Non Patent Literature 5); InternationalPublication No. WO 2003/002142; International Publication No. WO2003/028757; International Publication No. WO 2003/028758; InternationalPublication No. WO 2008/105462; U.S. Pat. No. 7,030,212; U.S. PatentApplication Publication No. 2004/097703; U.S. Patent ApplicationPublication No. 2007/082860; U.S. Patent Application Publication No.2006/205667; U.S. Patent Application Publication No. 2006/217297; U.S.Patent Application Publication No. 2008/152631; U.S. Patent ApplicationPublication No. 2010/292160; U.S. Patent Application Publication No.2011/098233; U.S. Patent Application Publication No. 2011/070251; U.S.Patent No. 2010/062010; U.S. Pat. No. 7,063,854; U.S. Patent ApplicationPublication No. 2010/247556; U.S. Patent Application Publication No.2008/070835; U.S. Patent Application Publication No. 2006/165708; U.S.Patent Application Publication No. 2004/247609; U.S. Patent ApplicationPublication No. 2005/002951; and U.S. Patent Application Publication No.2010/247556 (all are incorporated herein by reference).

Among others, preferred is a peptide selected from the group consistingof:

(SEQ ID NO. 2) Cys Tyr Thr Trp Asn Gln Met Asn Leu; (SEQ ID NO. 3)Arg Met Phe Pro Asn Ala Pro Tyr Leu; (SEQ ID NO. 4)Tyr Met Phe Pro Asn Ala Pro Tyr Leu; (SEQ ID NO. 5)Ser Leu Gly Glu Gin Gln Tyr Ser Val;and

A peptide may be used which consists of an amino acid sequence in whichone or several, preferably one or two amino acids are substituted,deleted, and/or added in the amino acid sequence of each of thesepeptides and has an function as the CTL peptide or the helper peptide.

The dose of the WT1 peptide is properly varied depending on the age andsex of a patient, the severity of disease, and the like; however, it istypically 0.0001 mg to 1,000 mg, preferably 0.01 mg to 100 mg, morepreferably 0.01 mg to 10 mg, for example 1, 2, 3, or 5 mg peradministration.

Examples of the dosage form suitable for the administration of the WT1peptide include a water-in-oil (w/o) emulsion, an oil-in-water (o/w)emulsion, a water-in-oil-in-water (w/o/w) emulsion, a liposomeformulation, a microsphere formulation, a microencapsulated formulation,a solid injection, or a liquid formulation. These formulations may belyophilized formulations. The formulation may contain, as needed, astabilizer (for example, a polysaccharide, an amino acid, a protein,urea, or sodium chloride), an excipient (for example, a saccharide, anamino acid, urea, or sodium chloride), an antioxidant, a preservative,an isotonizing agent, or a buffering agent. The formulation foradministering the WT1 peptide may be formulated in advance or may beprepared at the time of use in administration to a patient. The WT1peptide may be administered together with an appropriate adjuvant or maybe formulated together with an adjuvant.

Temozolomide is an anti-cancer agent as a monovalent alkylating agent,is non-enzymatically converted to methyldiazonium ion under conditionsof physiological pH, and exhibits an anti-tumor effect by methylatingDNA. According to the method of the present invention, the dosage formof temozolomide and the method of administration thereof are notparticularly limited. Temozolomide is sold in the forms of capsules andintravenous drip infusions under the trade name “Temodar (R)” fromSchering-Plough Corporation; according to the present invention, thecapsule may be orally administered, or the injection may beintravenously administered. The dose of temozolomide is properly varieddepending on the age and sex of a patient, the severity of disease, andthe like; however, it is typically 50 to 300 mg/m² (body surface area),preferably 75 to 200 mg/m² (body surface area), for example, 75, 150, or200 mg/m² (body surface area) on a once-daily basis.

The period and time of administration of the WT1 peptide andtemozolomide are not particularly limited. The WT1 may be administeredfrom before the administration of temozolomide, may be administered overthe whole or partial period of administration of temozolomide, or may beadministered after the administration of temozolomide. For example, in astandard therapy for newly-diagnosed glioma, temozolomide is orallyadministered at 75 mg/m² (body surface area)/administration once dailyfor continuous 42 days in combination with radiation irradiation as anafter-treatment after extirpative surgery for tumor, followed by 4 weeksoff treatment. Thereafter, temozolomide alone is orally given at 150mg/m²/administration once daily for continuous 5 days, followed by 23days off treatment. The 28 days are defined as 1 course, and at thesecond course and later, temozolomide is orally given at 100, 150, or200 mg/m²/administration once daily for continuous 5 days, followed by23 days off treatment. In this case, after the end of radiation therapy,the WT1 peptide may be administered for the same period as the period ofadministration of temozolomide, may be administered for a portion of theperiod of administration of temozolomide, may be continuouslyadministered for a period including the duration of the off-treatment,or may be administered for a period other than the period ofadministration of temozolomide. The above treatment regimens are onlyillustrative, and the patient may receive or not receive otheranticancer therapies including radiation therapy or extirpative surgeryfor tumor.

According to the present invention, the treatment of cancer includesboth preventive treatment and therapeutic treatment. Examples of thetreatment of cancer include the shrinkage or suppression of increase oftumor lesions, the suppression of appearance of new lesions, theprolongation of survival, the improvement or suppression of progressionof subjective and objective symptoms associated with tumor, thesuppression of metastasis, and the prevention of recurrence.

The method of the present invention can be used for the treatment ofvarious cancers including hematopoietic organ tumors such as leukemia,myelodysplastic syndrome, multiple myeloma, and malignant lymphoma, andsolid cancers such as glioma, stomach cancer, large bowel cancer, lungcancer, breast cancer, germ-cell cancer, liver cancer, skin cancer,bladder cancer, prostate cancer, uterine cancer, uterocervical cancer,and ovary cancer; however, it is particularly suitable for glioma.

The present invention also relates to: a pharmaceutical compositioncomprising a WT1 peptide for treating cancer, wherein the composition isused in combination with temozolomide; a kit for treating cancer,comprising a WT1 peptide and temozolomide; and the use of a WT1 peptidefor treating cancer, wherein the WT1 peptide is used in combination withtemozolomide. The dosage form, dose, and the like of the WT1 peptide andtemozolomide for the pharmaceutical composition, kit, and use of thepresent invention are as described in the method for treating cancer.

The present invention will be specifically described below withreference to Examples. However, the present invention is not limited tothese Examples in any sense.

Example 1 1. Method

The phase I clinical trial of chemoimmunotherapy using temozolomide anda modified WT1 peptide (mp235-243) in combination as maintenance therapyafter postoperative adjuvant chemoradiation therapy was performed inpatients with newly-diagnosed malignant glioma.

(1) Test Agent

(i) WT1 Peptide Vaccine

Principal Ingredient: HLA-A*2402 restricted modified WT1 peptide(mp235-243): CYTWNQMNL (SEQ ID NO: 2)

Adjuvant: Montanide ISA 51 (250 μg/A): Freund's incomplete adjuvant

A saline solution of the WT1 peptide and Montanide ISA 51 were mixed ata weight ratio of 1:1 to prepare a water-in-oil emulsion, which was thenused as a WT1 peptide vaccine.

(ii) Temozolomide

Trade Name: Temodar (R)′ (Schering-Plough Corporation)

Dosage Form and Dose: Capsule 20 mg, 100 mg

(2) Patient

The patients were 5 HLA-A-2402 patients with glioblastoma and 1HLA-A-2402 patient with anaplastic astrocytoma in whom WT1 gene wasdemonstrated to be expressed in the tumors.

(3) Treatment Regimen

As an after-treatment after extirpative surgery for tumor, 5-day/week 2Gy radiation irradiation was performed once daily for 6 weeks andtemozolomide (75 mg/m² once daily) was orally daily administered inirradiating radiation for 6 weeks as a rule (49 days at the longest).From the first to second weeks after the end of theradiation/temozolomide combination therapy, WT1 peptide vaccine therapywas started and the WT1 peptide vaccine (3 mg) was administered atintervals of 1 week. From the fourth week after the end of the radiationtherapy, temozolomide (150 to 200 mg/m² once daily) was orally dailyadministered for 5 days. When the WT1 vaccine therapy was started fromthe first week after the end of the radiation/temozolomide combinationtherapy, the WT1 peptide vaccine was administered 3 times before theadministration of maintenance temozolomide and 4 times from the time ofthe administration of maintenance temozolomide, 7 times in total. Whenthe therapy was started at the second week, the number of times ofadministration was set to 6 in total.

(4) Evaluation

-   -   (i) Tumor Response

The tumor response was evaluated using RECIST criteria(http://www.jcog.jp/). Tumor measurable by MRI as a rule was defined asa measurable lesion and had a maximum diameter of 10 mm or more in thecase of a slice of 5 mm or less and a maximum diameter of 20 mm or morein the case of a slice of 5 mm to 10 mm (inclusive); however, it wasdetermined as measurable if the maximum diameter was 2 times or more theslice width. Up to 5 of the measurable lesions observed before the startof administration in the order of decreasing the maximum diameter(hereinafter, referred to as the major diameter) were defined as targetlesions, and lesions not chosen as the targets was all defined asnon-target lesions irrespective of whether being measurable or not.Evaluation was performed within 2 weeks before the start of therapy bythe administration of the WT1 peptide vaccine, every 4 weeks, as a rule,after the start thereof, and at the sixth week after the firstadministration of maintenance temozolomide.

(a) Response of Target Lesion

The response of target lesions was determined according to the followingcriteria.

TABLE 1 Response Evaluation Criteria for Target Lesion ClassificationDefinition CR Disappearance of all target lesions (Complete Response)including secondary changes due to tumor. PR A 30% or more decrease inthe sum of (Partial Response) the major diameter of target lesions ascompared to the sum of the major diameter thereof before treatment. PD A20% or more increase in the sum of (Progression the major diameter oftarget lesions Disease) (including recurrence), compared to the smallestsum of the major diameter thereof previously observed. However, anincrease in such a range that the absolute value of the sum of the majordiameter thereof does not exceed 10 mm is not defined as PD. SD Neithertumor shrinkage to qualify for (Stable Disease) PR nor tumor increase toqualify for PD. NE Incapability of examination for any (Not Evaluable)reason, or incapability of determination as CR, PR, PD, or SD.

Percentage of reduction of sum of major diameter=(Sum of major diameterbefore therapy−Sum of major diameter at evaluation)/Sum of majordiameter before therapy×100%

Percentage of increase of sum of major diameter=(Sum of major diameterat evaluation−Smallest sum of major diameter)/Smallest sum of majordiameter×100%

(b) Response of Non-Target Lesion

The response of non-target lesions was determined according to thefollowing criteria.

TABLE 2 Response Evaluation Criteria for Non-Target LesionClassification Definition CR Disappearance of all non-target lesions.IR/SD No disappearance of one or more non-target (Incomplete lesions.Response/Stable Disease) PD Increase in non-target lesions (includingreccurrence). NE Incapability of examination for any reason, (NotEvaluable) or incapability of determination as CR, IR/SD, or PD.

(c) Overall Response

Overall response was determined according to the following from a tumorresponse for target lesions, a tumor response for non-target lesions,and the presence of appearance of a new lesion. When either targetlesions or non-target lesions were determined to be NE, overall responsewas NE.

TABLE 3 Evaluation Criteria for Overall Response Response for Presenceof Response for Non-target Appearance of Overall Target Lesion LesionNew Lesion Response CR CR No CR CR IR/SD No PR PR Other Than PD No PR SDOther Than PD No SD PD Not Questioned Not Questioned PD Not QuestionedPD Not Questioned PD Not Questioned Not Questioned Yes PD

(d) Best Overall Response

Overall response was defined as “better” in the order of CR, PR, SD, PD,and NE, and best overall response was determined from overall responsesfor all courses according to the following criteria. When the bestoverall response corresponded to the definitions of a plurality ofdivisions, it was divided into a better one in the order of CR, PR, SD,PD, and NE.

TABLE 4 Evaluation Criteria for Best Overall Response ClassificationDefinition CR Resulting in an overall response of CR continuously 2times or more at intervals of 4 weeks or more. PR Resulting in anoverall response of PR or higher (CR or PR) continuously 2 times or moreat intervals of 4 weeks or more. SD Resulting not in a best overallresponse of CR or PR but in the overall response being not PR at aninterval of at least 12 weeks after the start of therapy and the overallresponse being SD or higher once or more. PD Resulting in an overallresponse of PD without corresponding to a best overall response of CR,PR, or SD. NE Resulting in an overall response of NE in all courses.

(ii) Progression-Free Survival (PFS)

The day of surgery was used as the initial date of reckoning, and theperiod therefrom to the earlier date on which the disease was determinedto have progressed and the date of death due to any cause was defined asprogression-free survival (PFS).

2. Result

The results of evaluating the 6 patients are shown below.

TABLE 1 PFS OS Case Age/Sex Disease Name Response (weeks) (weeks) 142/Male Glioblastoma CR >84.6 >84.6 2 55/Female GlioblastomaCR >70.0 >70.0 3 48/Male Anaplastic CR >64.7 >64.7 Astrocytoma 4 41/MaleGlioblastoma CR >56.4 >56.4 5 45/Male Glioblastoma CR >33.0 >33.0 660/Male Glioblastoma PD 12.0 >33.0 PFS: Progression-free survival OS:Total survival

In 5 of the 6 tested patients, the evaluation of CR (complete response)was obtained and PFS (progression-free survival) was 30 weeks or more;in 4 patients, PFS was as long period as 50 weeks or more.

Example 2

In patients 1 and 3, therapy by the administration of the WT1 peptidevaccine (3 mg, once per month) was continued, and in patients 2, 4, and5, therapy was continued in which 1 course consisted of 28 days overwhich temozolomide was orally daily administered for 5 days (150 to 200mg/m², once daily) followed by 23 days off treatment and the WT1 peptidevaccine was administered (3 mg, once weekly) for 4 weeks from the startof the administration of temozolomide. Therapy was discontinued inpatient 6. As a result, the survival of all patients was prolonged, andPFS (progression-free survival) was 80 weeks or more in 5 of the 6patients and 100 weeks or more in 4 patients.

TABLE 2 PFS OS Case Age/Sex Disease Name Response (weeks) (weeks) 142/Male Glioblastoma CR >135.1 >135.1 2 55/Female GlioblastomaCR >120.5 >120.5 3 48/Male Anaplastic CR >115.2 >115.2 Astrocytoma 441/Male Glioblastoma CR >106.9 >106.9 5 45/Male GlioblastomaCR >83.5 >83.5 6 60/Male Glioblastoma PD 12.0 >85.3 PFS:Progression-free survival OS: Total survival

A comparison is shown in FIG. 1 between the total survival of patientsgiven only radiation therapy or only a combination therapy of radiationtherapy and temozolomide as reported in N. Engl. J. Med., 2005; 352:9870996 (incorporated herein by reference) and the total survival of thepatients given the combination therapy of the WT1 peptide vaccine of thepresent invention and temozolomide.

These results show that an excellent clinical effect is obtained by thecombined use of the WT1 peptide vaccine therapy and chemotherapy withtemozolomide.

SEQUENCE LISTING FREE TEXT SEQ ID NO. 2: Synthetic Peptide SEQ ID NO. 3:Synthetic Peptide SEQ ID NO. 4: Synthetic Peptide SEQ ID NO. 5:Synthetic Peptide

1. A method for treating cancer, comprising administeringtherapeutically effective doses of a WT1 peptide and temozolomide to apatient in need thereof.
 2. The method according to claim 1, wherein theWT1 peptide is a peptide selected from the group consisting of:(SEQ ID NO. 2) Cys Tyr Thr Trp Asn Gln Met Asn Leu; (SEQ ID NO. 3)Arg Met Phe Pro Asn Ala Pro Tyr Leu; (SEQ ID NO. 4)Tyr Met Phe Pro Asn Ala Pro Tyr Leu; (SEQ ID NO. 5)Ser Leu Gly Glu Gln Gln Tyr Ser Val;

and a peptide comprising one or two amino acid substitutions, deletionsor additions of SEQ ID Nos: 2, 3, 4, or
 5. 3-6. (canceled)
 7. The methodaccording to claim 1, wherein the WT1 peptide consists of Arg Met PhePro Asn Ala Pro Tyr Leu (SEQ ID NO. 3).
 8. The method according to claim1, wherein the WT1 peptide consists of Tyr Met Phe Pro Asn Ala Pro TyrLeu (SEQ ID NO. 4).
 9. The method according to claim 1, wherein the WT1peptide consists of Ser Leu Gly Glu Gln Gln Tyr Ser Val (SEQ ID NO. 5).10. The method according to claim 1, wherein the WT1 peptide is apeptide in which a cysteine residue is added at the N terminal end ofthe peptide selected from the group consisting of: (SEQ ID NO. 3)Arg Met Phe Pro Asn Ala Pro Tyr Leu; (SEQ ID NO. 4)Tyr Met Phe Pro Asn Ala Pro Tyr Leu;  and (SEQ ID NO. 5)Ser Leu Gly Glu Gln Gln Tyr Ser Val.


11. The method according to claim 10, wherein the cysteine is added bycondensing a thiol group of the cysteine and a thiol group of thecysteine residue in the peptide via disulfide bonding.
 12. The methodaccording to claim 1, wherein the WT1 peptide is a peptide dimer inwhich two peptide monomers are bonded together via disulfide bondingbetween thiol groups, and the two peptide monomers are selected from thegroup consisting of (SEQ ID NO. 2) Cys Tyr Thr Trp Asn Gln Met Asn Leu;(SEQ ID NO. 3) Arg Met Phe Pro Asn Ala Pro Tyr Leu; (SEQ ID NO. 4)Tyr Met Phe Pro Asn Ala Pro Tyr Leu; (SEQ ID NO. 5)Ser Leu Gly Glu Gln Gln Tyr Ser Val;

and a peptide comprising one or two amino acid substitutions, deletionsor additions of SEQ ID Nos: 2, 3, 4, or
 5. 13. The method according toclaim 12, wherein the peptide monomers are selected from the groupconsisting of: (SEQ ID NO. 2) Cys Tyr Thr Trp Asn Gln Met Asn Leu,

and a peptide in which a cysteine residue is added at the N terminal endof the peptide selected from the group consisting of: (SEQ ID NO. 3)Arg Met Phe Pro Asn Ala Pro Tyr Leu; (SEQ ID NO. 4)Tyr Met Phe Pro Asn Ala Pro Tyr Leu;  and (SEQ ID NO. 5)Ser Leu Gly Glu Gln Gln Tyr Ser Val.


14. The method according to claim 1, wherein the WT1 peptide is apeptide dimer of SEQ ID NO: 3, each monomer of SEQ ID NO:3 has acysteine at the N-terminal end of the peptide and is bonded together viadisulfide bonding between thiol groups of the cysteine.
 15. The methodaccording to claim 1, wherein the WT1 peptide is a peptide dimer of SEQID NO: 2 and SEQ ID NO:3, each of SEQ ID NO:2 and SEQ ID NO:3 has acysteine at the N-terminal end and bonded together via disulfide bondingbetween thiol groups of the cysteine on each of SEQ ID NO:2 and SEQ IDNO:3.
 16. The method according to claim 1, wherein the cancer is glioma.17. The method according to claim 1, wherein the WT1 peptide andtemozolomide are administered to a patient having received extirpativesurgery for a tumor.
 18. The method according to claim 1, wherein theWT1 peptide and temozolomide are administered to a patient havingreceived radiation therapy.
 19. The method according to claim 1, whereinthe WT1 peptide and temozolomide are administered to a patient havingreceived a combination therapy of radiation therapy and temozolomide.